Nematodes represent a group of unsegmented round worms. They are simple in anatomy, having a simple gut and elongated fusiform shape. They are divided into numerous Families, some which are free living while others are parasitic to plants or animals. Those which are parasitic to insects are called entomogenous or entomopathogenic nematodes.
The Order of greatest commercial interest for insect control is the Order Rhabditida, which contains several Families, many of whose members are parasitic to insects. Prominant among these Families are the Steinernematids and Heterorhabditids. A general discussion of the classification of nematodes, and the entomogenous Families thereof is found in Poinar, G.O., "The Natural History of Nematodes" (1983), Prentice-Hall, Inc., N.J.
Nematodes have a standard life cycle comprising five stages which are delineated by a molting process in which a new cuticle is formed and the old one shed. Briefly, the adults of stage 5 reproduce, and the eggs generate stage 1 larvae, which, under appropriate conditions, transit to stage 2. Normally, the stage 2 larvae simply develop to stage 3 larvae and thence to stage 4 larvae, which then complete the cycle to the adult stage. However, and of interest to the use of nematodes for insect control, when conditions are relatively unfavorable for continuing growth and reproduction, the stage 2 larvae of Steinernematid and Heterorhabditid nematodes develop instead into "stage 3 infective juveniles" or "IJs". Under these conditions, the cuticle characteristic of the second stage is retained and is called the sheath. It completely encloses the nematode. IJs are infective to insects and complete their life cycle through stage 4 and adult at the expense of the host.
Steinernematid and Heterorhabditid IJ nematodes are an effective means of insect control. They are identifiable morphologically and normally live in surface water films around soil particles. They require oxygen and moisture for survival, but do not feed; they utilize their own food reserves as an energy source. They remain infective if the sheath is removed.
One other aspect of Steinernematid and Heterorhabditid nematode biology is significant: nematodes within these families are symbiotic with species of bacteria which are primarily but not totally responsible for their entomopathogenic properties.
The commercial production of Steinernematid and Heterorhabditid nematodes and their use in insect pest control presents a number of challenges which have only recently begun to be met. Large scale production of IJs has been developed at a number of locations, and a number of techniques have been tried. See, for example, Soviet Patent 726,164; Apr. 8, 1980; PCT Patent Application No. 86/01074 published Feb. 27, 1986; U.S. Pat. No. 4,334,498 and U.S. Pat. No. 4,178,366.
Formulations have also been devised for the application of infective juveniles to the soil. See, for example, Soviet Patent Application No. 378,222 and U.S. patent application Ser. No. 4,178,366. One approach utilizes a suspension in light mineral oil. In addition, Japanese Patent Application No. 60/260,678 proposes a fermented compost support for the application of the nematodes.
An additional and serious problem in commercialization of insect control using Steinernematid and Heterorhabditid nematodes arises in the large scale shipment and storage of the infective juveniles in a state which maintains their viability and pathogenicity. Heretofore, relatively impractical methods, which only minimally reduce nematode metabolism have been used. These include storage and transportation in oxygenated water (Dutky, S. R., et al, J Insect Pathol (1964) 6:417-422) in sterile water or 0.1% formalin in flasks (Poinar, G. O., "Nematodes for Biological Control of Insects" (1975) CRC Press, Boca Raton, Fla.) or in 0.1% formalin on moist polyurethane sponge or saturated filter paper (Bedding, R.A. Ann Applied Biol (1984) 104:117-120; Hara, A. H. et al, USDA Adv Agric Technol W-16 (1981); Howell, J. F., J Invert Pathol (1979) 33:155-156 and Lindergren, J. E. at al, USDA Adv Agric Technol W-3 (1979)). Other shipment and storage techniques have included the use of wood chips and activated charcoal.
Recently, additional approaches have been disclosed. U.S. Pat. No. 4,417,545 describes a shipping and/or storage container for nematodes and/or their eggs in their dormant state. This container basically sandwiches the nematodes and eggs between two pieces of foam which are saturated with water and thus maintain a high level of humidity. This approach is however directed to the noninfective stages of the worm and does not relate to the shipment of infective juveniles. PCT Application WO85/03412 suggests methods of transport and storage which depend on maintaining putative anaerobic conditions and the presence of an antimicrobial agent. High osmotic strength solutions are also used to prevent bacterial growth. The proposed storage conditions also include an adsorbent such as charcoal or synthetic resins, although it is not clear what these agents are expected to adsorb. The disclosure exemplifies the use of formaldehyde as an antimicrobial, and proposes storage containers which contain both the nematodes and adsorbent charcoal.
The approach of the present invention is to maintain the infective juveniles of the Steinernematid and Heterorhabditid nematodes in a state of dormancy so that their food reserves are not used up, and so that upon return to suitable conditions they revive and remain pathogenic to the insect host. In short, the methods and containers disclosed in connection with the present invention are designed to maintain the infective juveniles in a "cryptobiotic" state--a state of dormancy in which metabolism is suppressed. Several ways of doing this, with varying degrees of success, are known for organisms in general. The most generally suggested method and perhaps the most universally applicable is the induction of cryobiosis, i.e., reduced metabolism at low, usually freezing temperatures. In addition, and more difficult to achieve, are anhydrobiosis, which is induced by evaporative desiccation and the closely related osmobiosis, which is induced by osmotic desiccation.
There is an extensive literature on anhydrobiosis in nematodes in general, although any detailed disclosure related to the nematodes of interest in insect control is limited to a single report (Simons, W. R., and Poinar, G. O., J Invert Pathol (1973) 22:228-230). An additional report that Neoaplectana desiccate in nature under unspecified conditions appears in a symposium abstract (Kamionek, M. et al, "Eleventh Int'l Symp Nematol. Eur Soc Nematol" (1972)).
Other types of nematodes, including free living and plant parasitic nematodes, are known to survive naturally under dry conditions (Evans, A.A.A.F. et al, in "Nematodes as Biological Models" (1980) Academic Press, New York, pp. 193-211; Demeure, Y. et al, in "Plant Parasitic Nematodes" (1981) Academic Press, New York). It has been shown that significant changes in chemical composition occur in preparation for the anhydrobiosis caused by desiccation, and it is known that the plant parasitic nematodes which form the subjects of these studies, must be preconditioned at 97-98% relative humidity for 48-72 hours before being subjected to lower relative humidity (Evans et al (supra); Womersley, C., Comp Biochem Physiol (1981) 68A:249-252; Madin, K.A.C., et al. J Expl Zool (1975) 143:335-342; Crowe, J. H., et al, (ibid) 323-334).
Freckman, D. W. et al, in "New Trends in Soil Biology" (Lebrun, P. ed.) (1983) Universities Catholique de Louvain Press, discuss the ability of nematodes in desert soils to survive anhydrobiosis. Womersley, C. Compar Biochem Physiol (1981) 70B: 669-678 reviews the mechanisms of anhydrobiotic survival in nematodes; similar studies are reported by Crowe, J. H., et al. J Exp Zool (1979) 207:431-437; Demeure, Y., et al. J Nematol (1979) 11:189-195 and Crowe, J. H., et al, Ann Meet Amer Inst Biol Sci, East Lansing, Mich. 21-26 August 1977.
However, with respect to species of interest in insect control, the one report of an attempt to desiccate N. carpocapsae (Simons, W. R., and Poinar, G. O., supra) utilized a series of humidity chambers containing glycerol solutions. Relative humidity (RH) was not measured directly, nor was the temperature at which the experiment was conducted reported. IJs were held at 96% RH for 12 hr, transferred to 93% for a further 12 hr, and then to RHs ranging from 10-79% for periods up to 28 days. Only at 79.5% RH was survival greater than 40% after 12 days; even under these conditions viability fell to 30% after 20 days.
The present invention is based on the findings that 1) a minimum period for induction of anhydrobiosis at high RH is required and that 2) Steinernematid and Heterorhabditid infective juveniles are extremely fastidious with respect to accurate and constant RH control. The latter point has relevance with respect to acceptable means for carrying out the invention and with regard to the interpretation of the literature. In particular, we have found that when glycerol is used to control RH, nematode survival after induction of anhydrobiosis is highly variable. Nematode survival data after induction of anhydrobiosis in air whose RH is controlled by sulfuric acid solutions, on the other hand, is highly consistent. We have directly measured the RH levels above glycerol solutions and found them to be unreliable and unpredictable for the precise control required. Far more consistent RH control is achieved with sulfuric acid solutions. Thus, the results of Simons and Poinar are difficult to interpret because glycerol does not offer a dependable means to control RH.
In short, none of the published studies of nematode desiccation provide guidance for effecting anhydrobiosis in Steinernematid and Heterorhabditid entomogenous nematodes in a scalable process to ensure effective, commercially practical, long-term mass storage and shipment.